Optimization of test conditions for development of MTT as in vitro screen.

ABSTRACT

The quick and easy method of tetrazolium based colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test the viability of the adult parasites of a rodent filariid Acanthocheilonema viteae in vitro. The ideal conditions required for antifilarial screening were determined by correlating the MTT reduction ability of worms with their size and age in the vertebrate host, also the duration of incubation and temperature of the in vitro culture. It was observed that the worms collected from the host after 90 days of L3 (infective larvae) exposure were not suitable for in vitro screen as they could not reduce MTT to that extent as the worms of early infection. Healthy and full grown worms and also those incubated at 37 degrees C for 16 hr or more caused maximum MTT reduction. Thus, it is recommended to select healthy adult filariids of proper age and size (male > 3.5 cm; female > 7.0 cm). The incubation temperature of the in vitro culture system needs to be adjusted to 37 degrees C and parasites might be exposed to drugs upto 24 hr without much alteration in MTT reduction of untreated controls.